Project Summary The long-term research objective is to design, synthesize and investigate model compound systems which can help elucidate fundamental aspects of structure, metal-ligation, spectroscopy and reactivity relevant to the dioxygen (O2) and nitrogen oxide (NOx) chemistry which occurs in heme-copper oxidases (e.g., CcOs), nitric oxide reductases (NORs) and related proteins. HCOs and NORs are evolutionarily related enzymes which play critical roles in cellular processes within aerobic and anaerobic organisms. They have a heme/M (M = Cu or non-heme Fe) active site that reductively cleaves O2 or couples NO, respectively. The research proposed will contribute to a better understanding of enzyme structure and mechanism by providing a comprehensive and fundamental basis relevant to biological processing of O2 and NO that extends even beyond the heme and Cu metalloprotein sphere. Specific aims include: (A) the study of O?O cleavage chemistry in low-spin heme-(?-1- 2)-peroxo-Cu complexes by addition of appropriate H+/e? sources, including phenols or derivatives. The large degree of synthetic variability in porphyrins and copper ligands, along with substrates of varying pKa's and/or E0, will be utilized in order to meticulously study the factors which lead to successful O2-activation and reductive cleavage (vs. other pathways) in the context of structure-function relationships of CcO & model systems. (B) the generation of new heme-peroxo-Cu complexes (with incorporated biomimetic or H-bonding moieties) and detailed characterization of their structural and electronic properties. (C) the elaboration of two chemical systems designed to test how CcO (bio)chemistry leads to the actual formation of the copper-ligand His-Tyr crosslink. Detailed studies will involve new new ligand scaffolds bearing open imidazole N-H sites poised for covalent coupling to an exogenous phenolic substrate. An oxidative element will be included, such as the presence of a peroxo group or high-valent iron-oxo species. (D) investigation of chemical systems with input variations of the heme axial `base' ligand, where heme/NO/O2 coordination chemistry will be studied mechanistically with regard to peroxynitrite formation and its subsequent decay or substrate reactivity. This chemistry occurs in NO dioxygenases, enzymes critically involved in cellular NO homeostasis (and cellular signaling) via amino-acid nitration chemistry. (E) the study of chemistry relevant to NORs, heme/Cu or heme/non-heme Fe assemblies that enable NO reductive coupling. A clear focus will be on the study of the formation of putative hyponitrite intermediates, their structures and their reactivity leading to N2O as product. Such information is key to the understanding of N-N coupling and N-O cleavage chemistries which also involve protonation events. These processes are of broad interest with respect to other biological metalloenzyme nitrogen oxide processing. In-hand heme/Cu assemblies also enable this chemistry and further mechanistic probing is necessary; these processes are critical to NO signaling and linked to cellular responses to changes in [O2] concentrations.